Preclinical development of complex processing systems for ex vivo culture-expanded lymphohematopoietic cells, with subsequent immunologic and/or genetic manipulation, have been carried out in collaboration with a number of NIH Institute investigators, and with Nexell, Inc.Preparation of CD8-depleted, culture-expanded lymphocytes: In collaboration with NIAID and NCHGR, the Cell Processing Section has applied this 10-day process to a clinical trial of human immunodeficiency virus (HIV) gene therapy in the syngeneic twin model. This trial was completed in June 1998, but data are being collected and analyzed with regard to the relative efficacy of the therapeutic and nontherapeutic vectors. In one patient not on antiretroviral therapy, the therapeutic vector-transduced cells had an in vivo survival advantage over the marker gene-transduced cells. Manuscripts on the methodology are in preparation or in press. The processing methods developed will be adapted to a new protocol using autologous mononuclear cells from HIV-infected patients in FY '99.Preparation of allogeneic donor lymphocytes selectively depleted for alloreactive T cells: This process is being developed, in collaboration with Dr. John Barrett and colleagues, for application to clinical allogeneic hematopoietic transplantation, especially in the HLA-mismatched setting. Over the past year, we have successfully developed a reliable method for expansion of selected T cells from patients with chronic myelogenous leukemia, resulting in a product that has minimal contamination with leukemia cells and is capable of stimulating a third-party (donor) immune response to recipient-specific antigens. In the next fiscal year, we will be adapting this process to the Aastrom bioreactor system, and using these stimulator T cells in an MLR, followed by selective depletion of the alloreactive donor cells by anti-CD25 Pseudomonas exotoxin.